Post-/co-translational modifications including phosphorylation, methylation, acylation, ubiquitination, SUMOlyation, and glycosylation play critical roles in determining the functions and fates of proteins (Walsh, C., ed. Posttranslational Modification of Proteins: Expanding Nature's Inventory. 2006, Roberts & Co: Englewood, Colo.). Among these modifications, glycosylation results in significant structural diversity and complexity of protein products. Usually, for the purpose of correlating glycosylation states with pathological changes, it is not a question of whether there is glycosylation, but rather the glycosylation pattern that marks protein function or different pathological states, including malignancy. For example, the glycosylation patterns of prostate specific antigen (PSA) from cancer cells in culture (Peracaula et al., Glycobiol., 2003. 13: 457-470) and prostate cancer patient's tissue and serum (Tabares et al., Glycobiology, 2006. 16(2): 132-145; Tabares et al., Clin. Biochem., 2007, 40: 343-350) are different from that of the normal prostate. Human pancreatic RNase 1, a glycoprotein secreted mostly by pancreatic cells, has completely different oligosaccharide chains when produced from pancreatic tumor cells, and deviation from the normal glycosylation pattern on fibrinogen, a protein critical to blood coagulation, can lead to coagulation disorders (Cohn et al., Pediatrics, 2006. 118: 514-521; Langer et al., J. Biol. Chem., 1988. 263: 15056-15063; Gilman et al., J. Biol. Chem., 1984. 259: 3248-3253; Hamano et al., Blood, 2004. 103: 3045-3050; Mirshahi et al., Thromb. Res., 1987. 48: 279-289; Ridgway et al., Br. J. Haematol., 1997. 99: 562-569; Rybarczyk et al., Cancer Res., 2000. 60: 2033-2039; Sugo et al., Blood, 1999. 94: 3806-3813). Pregnancy-related human chorionic gonadotropin (hCG) can provide biomarkers for cancer, Down syndrome, and pregnancy failure depending on its glycosylation patterns (Wang et al., Curr. Org. Chem., 2002. 6: 1285-1317; Gao et al., Org. Lett., 2003. 5: 4615-4618); and specific glycosylation patterns of haptoglobin (Hp) and alpha-fetoprotein (AFP) have a much higher degree of correlation with cancer than the total Hp/AFP levels (Yang et al., Chem. Biol., 2004. 11: 439-448).
Since certain glycoforms of these proteins are directly disease related, the ability to analyze and differentiate variations of glycosylation patterns in a given glycoprotein would be of value for the development of new diagnostics and biomedical research tools. Currently available analytical tools used for glycomics analysis include such as mass spectrometry, chromatography, especially capillary electrophoresis, antibody-based approaches, lectin profiling, and the like. However, there remains a need for techniques suitable for the rapid and accurate detection of protein glycosylation variations. Mass spectrometry and chromatography methods are time-consuming. Lectin profiling is useful for broad category glycan characterizations, but it only focuses on the glycan portion and does not give any indication as to the identity of the protein in question. As a result, purified or partially purified glycoproteins are usually needed for lectin-based characterizations in detail. Furthermore, cross-reactivity and low affinity are issues that may impede the application of lectins for highly specific characterizations. In addition, there are only about forty readily available lectins, which cannot satisfy the need for highly specific recognitions of various glycosylation patterns.
Molecules that can recognize a target glycoprotein with high affinity and specificity should preferably recognize both the glycan and the protein portions to be useful for glycoform-specific detection. However, antibodies and aptamer selection for the development of molecules of high specificity and affinity for glycoproteins do not have the intrinsic ability to specifically focus on the glycosylation sites in its native form, and allow for the ready differentiation of glycosylation variations.
Aptamer selection is a very powerful method for the development of custom-made nucleic acid-based high affinity “binders” (aptamers) for molecules of interest. Since the beginning of this field, a large number of aptamers have been reported for various applications with some in clinical trials or approved for clinical use. As powerful as the method is, aptamer selection has limited intrinsic ability to selectively focus on certain substructures of a large biomacromolecule. Therefore, methods for selection of aptamers that can recognize a glycoprotein and be able to differentiate its glycosylation patterns will be advantageous for the development of novel types of diagnostics and therapeutics as well as analytical tools for biomedical research.